Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion.

Bhuiyan NH, Selvaraj G, Wei Y, King J - J. Exp. Bot. (2008)

Bottom Line:
Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately.Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites.These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

ABSTRACTCell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

fig3: RT-PCR analysis of tissue-specific expression of monolignol biosynthesis in response to Blumeria graminis f.sp. tritici infection. TmSAMS1 (Bhuiyan et al., 2007) and TmPRX1 (Liu et al., 2005) genes were tested for comparison with the expression pattern of monolignol genes. Total RNA was isolated from epidermal (E) and mesophyll (M) tissues at 0 hpi and 24 hpi and reverse transcribed to cDNA. The expression of a chlorophyll a/b binding protein (TmCab) and a glyceraldehydes 3-phosphate dehydrogenase gene (TmGAPD) was used as an indicator of mesophyll contamination in epidermal tissues and a control of mRNA normalization, respectively.

Mentions:
The epidermis provides the major line of defence against infection by the surface-growing powdery mildew fungus. To investigate further whether the expression patterns of the monolignol biosynthesis genes differ between epidermis and other cell types, samples from susceptible line epidermis and mesophyll tissues were collected at 0 hpi and 24 hpi and amplified by RT-PCR using gene-specific primers. One marker gene, Cab (Chlorophyll a/b binding protein) was tested as an indicator of mesophyll contamination in epidermal tissues. One methyl cycle gene (TmSAMS1) and one peroxidase gene (TmPRX1) were also tested for comparison with monolignol genes. Distinct expression patterns among monolignol genes were found in the semi-quantitative RT-PCR analysis (Fig. 3).

fig3: RT-PCR analysis of tissue-specific expression of monolignol biosynthesis in response to Blumeria graminis f.sp. tritici infection. TmSAMS1 (Bhuiyan et al., 2007) and TmPRX1 (Liu et al., 2005) genes were tested for comparison with the expression pattern of monolignol genes. Total RNA was isolated from epidermal (E) and mesophyll (M) tissues at 0 hpi and 24 hpi and reverse transcribed to cDNA. The expression of a chlorophyll a/b binding protein (TmCab) and a glyceraldehydes 3-phosphate dehydrogenase gene (TmGAPD) was used as an indicator of mesophyll contamination in epidermal tissues and a control of mRNA normalization, respectively.

Mentions:
The epidermis provides the major line of defence against infection by the surface-growing powdery mildew fungus. To investigate further whether the expression patterns of the monolignol biosynthesis genes differ between epidermis and other cell types, samples from susceptible line epidermis and mesophyll tissues were collected at 0 hpi and 24 hpi and amplified by RT-PCR using gene-specific primers. One marker gene, Cab (Chlorophyll a/b binding protein) was tested as an indicator of mesophyll contamination in epidermal tissues. One methyl cycle gene (TmSAMS1) and one peroxidase gene (TmPRX1) were also tested for comparison with monolignol genes. Distinct expression patterns among monolignol genes were found in the semi-quantitative RT-PCR analysis (Fig. 3).

Bottom Line:
Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately.Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites.These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

ABSTRACTCell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.